小穗（spikelet）和小花（floret）是麥類作物花器官的基本結構，它們的發育直接影響到穗粒數和最終的籽粒產量。最近，Development雜誌在線發表了Jorge Dubcovsky實驗室（UC Davis）在小麥穗發育和形態建成方面的研究成果（doi:10.1242/dev.175398）。該研究系統解析了三個關鍵基因VRN1、FUL2和FUL3在小麥穗發育過程中的分子機理，並為將來提高小麥籽粒產量提供了直接證據。相關研究非常系統、全面，是一篇高質量的研究論文。我們有幸邀請到文章的一作--李成霞（Chenxia Li）老師進行解讀，小麥研究聯盟進行了翻譯（由於我們在小麥穗發育方面的知識有所欠缺，如出現錯誤請參考李老師的英文解讀）。
Wheat VRN1, FUL2 and FUL3 play critical and redundant roles in spikelet development and spike determinacy
小麥VRN1、FUL2和FUL3在小穗發育和穗部形態建成過程中起關鍵並冗餘作用
Chengxia Li(#), Huiqiong Lin(#), Andrew Chen, Meiyee Lau, Judy Jernstedt and Jorge Dubcovsky(*)
(*)：jdubcovsky@ucdavis.edu. Phone: 530 752 5159
The grass family (Poaceae) has approximately 10,000 species, including important food crops such as rice, maize, sorghum, barley and wheat.The flowers of these species are organized in a unique and diagnostic structure called spikelet, which is a compact inflorescence developing within the larger inflorescence. Spikelet is the basic unit of the grass inflorescence. Grass inflorescences have been described as a progressive acquisition of different meristem identities that begins with the transition of the vegetative shoot apical meristem (SAM) to an inflorescence meristem (IM). In wheat, the transition from vegetative SAM to IM is marked by the formation of a double-ridge structure, in which the lower leaf ridges are suppressed and the upper ridges acquire spikelet meristem (SM) identity andform spikelets. The number of spikelets per spike in wheat is determined by the number of lateral meristems formed before the transition of the IM into a SM toform the terminal spikelet. The growth of each wheatspikelet is indeterminate, with each SM initiating a variable number of floralmeristems (FM). The numbers of spikelets per spike and florets per spikelet determine the maximum number of grains per spike, therefore are important components of wheat grain yield potential.
禾本科（Poaceae）大約包含10,000個物種，其中包括水稻、玉米、高粱、小麥和大麥等重要的糧食作物。禾本科作物的花通過發育逐漸形成一種獨特的結構--小穗，該結構屬於一種在較大花序內發育的緊密花序。小穗是禾本科植物花器官的基本單位，其發育開始於營養生長的頂端分生組織（SAM）並逐漸向生殖生長的花序分生組織（IM）轉變。在小麥中，從SAM到IM的轉變以雙脊結構的形成為特徵，下部葉脊的發育受到抑制的同時上部脊繼續發育形成小穗分生組織（SM）並逐漸發育成小穗。在這個過程中，花序分生組織（IM）逐漸轉變為小穗分生組織（SM）並完成小穗的分化。在末端（最後一個）小穗分化形成之前所產生的側向分生組織的數量決定了小麥中每個穗子的小穗數。小麥每個小穗分生組織（SM）可分化成可變數量的花分生組織（FM），因此每個小穗的小花數並不固定。每個穗的小穗數和每個小穗的小花數決定了每個穗的最大穗粒數，是小麥籽粒產量潛力的重要組成部分。
In this study, we show that wheat MADS-box genes VRN1, FUL2 and FUL3 play critical and redundant roles in spikelet and spike development, and also affect flowering time and plant height. We combined loss-of-function mutants for the two homeologs of VRN1, FUL2 and FUL3 to generate double- and triple-null mutants in tetraploid wheat. In the vrn1ful2ful3-null triple mutant, the inflorescence meristem formed a normal double-ridge structure, however, itslateral meristems then proceeded to generate vegetative tillers subtended by leaves instead of spikelets. These results suggest an essential role of these three genes in the fate of the upper spikelet ridge and the suppression of the lower leaf ridge. Inflorescence meristems of vrn1ful2ful3-null and vrn1ful2-null remained indeterminate, and single vrn1-null and ful2-nullmutants showed delayed formation of the terminal spikelet and increased number of spikelets per spike. Moreover, the ful2-null mutant producedmore florets per spikelet, which together with a higher number of spikelets, resulted in a significant increase in the number of grains per spike in the field. Our results suggest that a better understanding of the mechanisms underlying wheat spikelet and spike development can inform future strategies toimprove grain yield in wheat.
我們的研究表明，小麥MADS-box基因VRN1、FUL2和FUL3在穗發育中起著關鍵和冗餘的作用，並且還影響開花時間和植株高度。我們分別利用VRN1、FUL2和FUL3在AB亞組同時突變的功能喪失突變體，在四倍體小麥中創製了雙重（4個基因）和三重（6個基因）突變體。在vrn1ful2ful3三重突變體中，花序分生組織能形成正常的雙脊結構，但其側向分生組織不能正常分化形成小穗，而是發育形成由葉子而不是小穗包圍的營養分櫱。這個結果表明VRN1、FUL2和FUL3在促進上部小穗脊的正常發育和抑制下部葉脊的發育過程中具有重要作用。vrn1ful2ful3三重突變體和vrn1ful2雙重突變體的花序分生組織發育異常，vrn1和ful2單突變體最後一個小穗原基分化較晚並導致了小穗數的增加。此外，ful2單突變體每個小穗還能產生更多的小花，並最終導致了穗粒數的增加。我們的研究結果闡明了小麥穗發育特別是小穗發育的分子機制，並為提高小麥穗粒數和產量提供了理論依據。
1. VRN1, FUL2 and FUL3 loss-of-function mutations reducestem elongation and delay flowering time
1. VRN1、FUL2和FUL3功能喪失突變體減少了莖的伸長並延遲開花
Plants carrying only the ful3-nullmutation showed no significant reduction in stem length, but those carrying thevrn1-null or ful2-null mutations were 20% and 14% shorter than the control, respectively (Fig. 1A). A three-way factorial ANOVA for stem length revealed highly significant effects for all three genes and significant synergistic interactions (Fig. 1C), indicating that VRN1, FUL2 and FUL3 have redundant roles in the regulation of stem elongation, and that the effect of the individual genes is larger in the absence of the otherparalogs.
ful3單突變體株系的莖長或株高並沒有明顯變化，但vrn1或ful2單突變的株系分別比對照矮20％和14％（圖1A）。三因素方差分析表明VRN1、FUL2和FUL3三個基因對莖杆長度具有顯著影響並存在明顯的協同作用（圖1C），這表明三個基因在莖杆伸長的調節中作用冗餘，並且在旁系同源基因缺失的情況下，單個基因的作用更大。
Functional redundancy among VRN1, FUL2 and FUL3 was also observed for heading time. The vrn1-null mutant headed 37.5 dlater than the control (Fig. 1D), but differences in heading time for the ful2-null, ful3-null and ful2ful3-null mutants in the presence of the strong Vrn-A1 allele were non-significant (Fig. 1E). For the vrn1ful2-null and vrn1ful2ful3-null mutants, it was not possible to determine heading times accurately because theyhad short stems and abnormal spikes that interfere with normal ear emergence. Instead, we determined the final number of leaves (Fig. 1B) and the timing of the transition between the vegetative and double-ridge stages (Fig. S3). The vrn1-null mutant had on average 14.4 leaves (59% > control, Fig. 1B), which was consistent with its later heading time (Fig. 1D). Similar leaf numbers were detected in vrn1ful2-null (14.3) and vrn1ful3-null (14.9), but the triple vrn1ful2ful3-null mutant had on average17.7 leaves (Fig. 1B), which was consistent with the 9 to 12 d delay in the transition between the vegetative SAM and the double-ridge stage relative to the vrn1-null control (Fig. S3).
對抽穗時間來說，VRN1、FUL2和FUL3三個基因同樣存在功能冗餘。vrn1突變體的抽穗期比對照晚37.5天（圖1D），但在強Vrn-A1等位基因存在下，ful2、ful3單突變體和ful2ful3雙重突變體的抽穗時間差異並不顯著（圖1E）。對於vrn1ful2雙重突變體和vrn1ful2ful3三重突變體來說，我們並不能準確地檢測其抽穗時間，因為這類突變體的莖非常短、穗部發育異常並且不能形成正常葉耳。但我們仍然可以統計葉片的最終數量（圖1B）以及小穗發育過程中從營養生長到生殖生長（雙脊期）過渡時間（圖S3）。vrn1單突變體平均具有14.4葉（比對照多59%，圖1B），其抽穗時間也相應地延長（圖1D）。在vrn1ful2雙重突變體（14.3葉）和vrn1ful3雙重突變體（14.9葉）中檢測到相似的葉片數，但是三重突變體vrn1ful2ful3平均具有17.7葉（圖1B），相應地三重突變體與vrn1單突變體相比開花時間也產生了9-12天的延遲（圖S3）。
Fig. 1. VRN1、FUL2和FUL3對莖長、葉片數目和抽穗時間的影響
2. VRN1, FUL2 and FUL3 play critical and redundant roles in spikelet development
2. VRN1、FUL2和FUL3在小穗發育過程中發揮著關鍵和冗餘的作用
Plants with individual vrn1-null,ful2-null and ful3-null mutations produced normal spikelets and flowers, but vrn1ful2-null or vrn1ful2ful3-null mutants had spike-like structures in which alllateral spikelets were replaced by leafy shoots (inflorescence tillers, Fig.2A-J). Removal of these inflorescence tillers revealed a thicker and shorter rachis with fewer internodes of variable length, but still retaining the characteristic alternating internode angles typical of a wild type rachis (Fig. 2B).
vrn1、ful2和ful3單突變的植物能產生正常的小穗和小花， vrn1ful2雙重或vrn1ful2ful3三重突變體雖能產生類似小穗的結構，但其所有的側面小穗均被葉狀枝條替代（花序分櫱，圖 2A-J）。去除這些花序分櫱後則顯示出較粗和較短的穗軸，節間變少、節間長度也有不同程度的變化，但仍然保留了典型的野生型交替的穗軸結構（圖2B）。
In vrn1ful2-null, approximately 70% of the central inflorescence tillers had leafy glumes, lemmasand paleas and abnormal floral organs, whereas the rest were fully vegetative. Floral abnormalities included leafy lodicules, reduced number of anthers, anthers fused to ovaries, and multiple ovaries (Fig. 2E-G). After the first modified floret, meristems from these inflorescence tillers developed two to five true leaves before transitioning again to an IM generating lateral VMs(Fig. 2E). The presence of both floral organs and leaves suggests that the originating meristem had an intermediate identity between VM and SM before transitioning to an IM. In the vrn1ful2-null double mutant the inflorescence tillers were subtended by bracts (Fig. 2C-D).
在vrn1ful2雙突變體中，大約70％的中央花序分櫱具有葉狀穎片、外稃和內稃以及異常的花器官結構，而其餘部分則全為營養器官。花的異常包括葉片化的漿片、花葯數量的減少、形成與子房融合的花葯和多個子房等（圖2E-G）。在第一個小花發育之後，來自花序分櫱的分生組織緊接著發育形成2至5個真葉，隨後再繼續發育形成側面的頂端分生組織（圖2E）。花器官和葉子的存在表明，早期的頂端分生組織在轉變為花序分生組織之前既可以向營養生長分化也可以向生殖生長分化。在vrn1ful2雙重突變體中，花序分櫱被苞片包圍（圖2C-D）。
In vrn1ful2ful3-null, the lateral meristems generated inflorescence tillers that had no floralorgans, and that were subtended by leaves in the basal positions and bracts in more distal positions (Fig. 2H-J). The presence of well-developed axillary tillers in these basal inflorescence leaves (Fig. 2H, L19 and L20) marked the border of the spike-like structure, because no axillary tillers or developing buds were detected in the true leaves located below this border (Fig. 2H,L11-L18).
在vrn1ful2ful3三突變體中，側生分生組織產生的花序分櫱沒有花器官，而是在基部位置產生葉片組織，在另一端產生苞片（圖2H-J）。在這些基部花序葉片組織中存在發育良好的腋生分櫱（圖2H，L19和L20），代表著穗狀結構的邊界，因為在該邊界下方的真葉中未檢測到腋生分櫱或發育中的芽（圖2H，L11-L18）。
Fig.2. vrn1ful2 雙重突變體和vrn1ful2ful3 三重突變體的表型變異
Scanning Electron-Microscopy (SEM) images of the early developing inflorescences in the vrn1ful2-null and vrn1ful2ful3-null mutants revealed elongated double-ridge structures similar to those in Kronos (Fig. 3 A) or vrn1-null (Fig. 3 C). Suppression of the lower leaf ridge was complete in Kronos (Fig. 3A) and in vrn1-null (Fig. 3D, red arrows), but was incomplete in vrn1ful2-null(Fig. 3B, E; yellow arrows), and even weaker in vrn1ful2ful3-null (Fig. 3C, F: green arrows). As a result of this change, inflorescence tillers were subtended by bracts in vrn1ful2-null (Fig. 2C-D) and by leaves in vrn1ful2ful3-null (Fig. 2H-I). The upper ridges (Fig. 3A-C, dots) transitioned into normal SMs in vrn1-null, with glume and lemma primordia (Fig. 3D, G), but looked like typical vegetative meristems in vrn1ful2-null and vrn1ful2ful3-null (Fig. 3E-F, H-I).
通過掃描電子顯微鏡觀察發現，vrn1ful2雙重和vrn1ful2ful3三重突變體中在發育早期的花序中出現了類似於野生型Kronos（圖3A）或vrn1單突變體（圖3C）中的細長雙脊結構。在Kronos（圖3A）和vrn1單突變體中（圖3D，紅色箭頭）下部葉脊的發育受到抑制，但在vrn1ful2雙重突變體（圖3B，E;黃色箭頭）和vrn1ful2ful3三重突變體中（圖3C，F：綠色箭頭）葉脊的發育並沒有受到正常抑制。因此，在vrn1ful2雙重突變體中花序分櫱被苞片替代（圖2C-D），而在vrn1ful2ful3三重突變體中花序分櫱被葉片替代（圖2H-I）。在vrn1單突變體中，上部脊（圖3A-C，紅、黃或綠點）轉變為正常的小穗分生組織（SM），並發育出穎片和外稃原基（圖3D，G），但在vrn1ful2雙重和vrn1ful2ful3三重突變體中則發育出了典型頂端分生組織（圖 3E-F，HI）。
Fig.3. 不同突變體的掃描電鏡觀察
3. FUL2and VRN1 haveredundant roles on spike determinacy and regulate the number of spikelets perspike
3. FUL2和VRN1在穗形態建成過程中存在功能互補並調節小穗數
Normal wheat spikes are determinate, with the distal IM transitioning into a terminal spikelet after producing arelatively stable number of lateral meristems (Fig. 4A). In vrn1ful2-null, by contrast, the IM was indeterminate (Fig. 4B) and continued to produce lateral meristems while growing conditions were favorable and eventually died without producing anyterminal structure. In the ful2-null background, one functional copy of VRN1 inthe heterozygous state was sufficient to generate a determinate spike (Fig.S6D, ful2-null/vrn-A1-nullvrn-B1), and the same was true for a single functional copy of FUL2 in a vrn1-null background (Fig. S6K, vrn1-null/ful2-A Ful2-B).
正常小麥在產生相對穩定數量的側分生組織後，遠端花序分生組織（distal IM）轉變為末端（最後一個）小穗（圖4A）。相比之下，在vrn1ful2雙重突變體中，遠端花序分生組織（IM）是不確定的（圖4B），在合適的生長條件下可繼續分化出側向分生組織，並且最終死亡。在ful2單突變體背景中，雜合狀態下VRN1的一個功能拷貝足以產生正常的幼穗（圖S6D，ful2 /vrn-A1 VRN-B1）；同樣的，在vrn1單突變體背景中，雜合狀態下FUL2的一個功能拷貝也能產生正常的幼穗（圖S6K，vrn1/ful2-AFUL2-B）。
The individual vrn1-nulland ful2-null homozygous mutantsshowed a larger number of spikelets per spike than the control. This increasewas 58% in the vrn1-null mutant (P< 0.0001, Fig. 4C) and 10% in the ful2-null mutant (P =0.0014, Fig 4D). Although no significant increases in the number of spikelets per spike were detected in the ful3-null mutant (P = 0.4096, Fig. 4E), two independent transgenic lines overexpressing FUL3 (Ubi::FUL3) showed an average reduction of 1.12 spikelet per spike relative to their non-transgenic sister lines (P = 0.0132 and P < 0.0001, Fig. S8A), which indicates that FUL3 can still play a role on the timing of the transition from IM to terminal spikelet.
與對照相比，vrn1或ful2單純合突變體每個穗子的小穗數增多：vrn1單突變體（P <0.0001，圖4C）的小穗數增加了58％，而ful2單突變體中的小穗數則增加了10％（P = 0.0014，圖4D）。儘管在ful3單突變體中小穗數沒有明顯增加（P = 0.4096，圖4E），但與非轉基因姊妹系相比，過表達FUL3的兩個獨立轉基因系（Ubi ::FUL3）的小穗數平均減少了1.12個（P = 0.0132和P <0.0001，圖S8A），這表明FUL3仍然可以在花序分生組織（IM）轉變到末端小穗的過程中發揮作用。
4. The ful2-null mutant produces a higher number of florets per spikelet andmore grains per spike in the field
4. ful2單突變體小花數量變多並最終導致大田環境中穗粒數的增加
In addition to the higher number of spikelets per spike, the ful2-null mutant produced a higher number of florets per spikelet than the Kronos control, an effect that was not observed for vrn1-null (Fig. 2A) or ful3-null(Fig. S10A). In spite of some heterogeneity in the distribution of spikelets with extra florets among spikes, the differences between the control and the ful2-null mutants were significant at all spike positions (Fig. S10B).
與Kronos野生型對照相比，ful2單突變體除了小穗數量較多外，每個小穗的產生的小花數量也明顯增加，這種變化在vrn1（圖2A）或ful3單突變體中（圖S10A）並未觀察到。儘管具有額外小花的小穗在整個穗子中的分布存在一些異質性，但對照和ful2單突變體之間的差異在所有穗位置都是顯著的（圖S10B）。
Based on its positive effect on the number of florets perspikelet and spikelets per spike (and its small effect on heading time), we selected the ful2-null mutant for evaluation in a replicated field experiment. Relative to the control, the ful2-null mutant produced 20% more spikelets per spike (P = 0.0002) and 9% more grains per spikelet (P= 0.05), which resulted in a 31% increase in the number of grains per spike (P = 0.0002, Fig. 4F). Although part of the positive effect on grain yield was offset by a 19% reduction in averagekernel weight (P = 0.0012), we observed a slight net increase of 6% in total grain weight per spike (P = 0.09, Fig. 4F). This negative correlation between grain number and grain weight suggests that in this particular genotype by environment combination grain yield was more limited by the 「source」 (produced and transported starch) than by the 「sink」 (number and size of grains).
基於ful2單突變體對小穗和小花數量的正面影響（對抽穗時間的影響較小），我們選擇了該突變體用于田間試驗中進行評估。於對照相比，ful2突變體每個穗的小穗數增加了20％的（P = 0.0002），每個小穗的穗粒數則增加了9％（P = 0.05），從而最終使穗粒數增加了31％（ P = 0.0002，圖4F）。雖然千粒重減少了19％（P = 0.0012），但我們仍發現每個穗子的總粒重量淨增加了6％（P = 0.09，圖4F）。穗粒數和千粒重之間的這種負相關性表明，在這種特定的基因型中，單株最終產量更容易受「源」（生產和運輸的澱粉）的限制，而不是穗粒數或籽粒大小的限制。
Fig.4.FUL2和VRN1在穗形態建成過程中存在功能互補並調節小穗數